Cre and other recombinase enzymes of the λ integrase family have opened new possibilities for the genetic manipulation of mammalian genomes (Kilby et al., 1993). These recombinases each cleave DNA at a specific target sequence and ligate the newly exposed ends to the cleaved DNA at a second target sequence. One member of this family, cre recombinase, the 38 kDa product of the bacteriophage P1 cre gene, catalyzes recombination between target sequences termed lox sites (Sternberg and Hamilton, 1981). The lox site is a 34 base pair target sequence consisting of two 13 base pair inverted repeats flanking an 8 base pair core (Hoess et al., 1982). When two lox sites are placed in cis, deletion or inversion of the DNA sequence between the lox sites ensues upon expression of cre, depending on the orientation of the lox sites with respect to each other. When lox sites are arrayed on two separate linear DNA molecules, strand exchange can occur; when present on a circle and a linear molecule, integration of the circle into the linear DNA can result from the action of. The ability to catalyze these reactions efficiently in mammalian cells (Sauer and Henderson, 1988) has enabled complex genetic manipulations of a sort only previously attainable in yeast and prokaryotes.
Of the λ integrase family members, the cre recombinase has become the most commonly employed site-specific recombinase for genetic manipulation, and conditional gene targeting in the mouse is its most frequent use (See Nagy, 2000; Muller, 1999; Rossant and McMahon, 1999; Sauer, 1998; Rajewsky et al., 1996; Kilby et al., 1993). Generally, a segment of a gene to be inactivated or modified by cre-mediated recombination is flanked by lox sites (“floxed allele”); this modification of an endogenous allele is accomplished by homologous recombination in ES cells. Being short sequences, strategically placed lox sites generally do not perturb expression of a target gene. A mouse generated from ES cells harboring a floxed allele can be bred to a mouse transgenic for the cre recombinase. Depending on how the transgenic cre gene is controlled, deletion of a floxed allele can take place in a tissue and/or in a developmentally precise manner, often bypassing lethality at earlier developmental stages or avoiding other unwanted effects.
Ideally, upon synthesis of cre, these cells should facilitate an analysis of the consequences of deleting a floxed allele(s) with precision. However, cre can be overtly toxic to cells, a property that inherently limits its utility. This toxicity depends upon the strand cleavage activity of cre, and is, therefore, intrinsic to its activity as a recombinase. A central feature of the toxicity is genomic instability. Other reasons for limiting the duration and intensity of recombinase expression exist as well, for example the potential antigenicity of a recombinase in a foreign host.
Thus, a need remains in the art for the creation of a recombinase system that eliminates recombinase-mediated toxicity or other undesired effects, but yet retains the ability to effect site-specific recombination.